The present invention relates to a method for high-resolution optical scanning of a sample, preferably using a laser scanning fluorescence microscope, the sample encompassing a substance that is shiftable into different energy states (first state Z1 and second state Z2), first state Z1 and second state Z2 differing from one another in terms of at least one optical property; the sample being illuminated, for local generation of first state Z1 of the substance, with light of a wavelength of the excitation spectrum of the substance; the sample being illuminated in a peripheral focus region of the excitation, for generation of second state Z2 of the substance, with light of a suitable de-excitation wavelength; and emitted light that proceeds from the sample and results from a decay of remaining first states Z1 being detected by means of a detection device.
The invention further relates to an apparatus for high-resolution optical scanning of a sample, in particular for carrying out a method according to one of claims 1 to 17, the sample encompassing a substance that is shiftable into different energy states (first state Z1 and second state Z2); first state Z1 and second state Z2 differing from one another in terms of at least one optical property; having at least one light source in order to illuminate the sample, for local generation of first state Z1 of the substance, with light of a wavelength of the excitation spectrum of the substance, and in order to illuminate the sample in a peripheral focus region of the excitation, for generation of second state Z2 of the substance, with light of a suitable de-excitation wavelength; and having a detection device for detecting emitted light that proceeds from the sample and results from a decay of remaining first states Z1.
Methods and apparatuses of the kind in question here have been known for some time from practical use and are used, for example, in the context of STED microscopy. With the imaging optical methods and apparatuses in question, it is possible to achieve spatial resolutions beyond the theoretical limit defined, in accordance with Abbe's law, by the diffraction limit that depends on the wavelength of the light that is used.
In the context of STED microscopy, a substance that is shiftable by light into an excited state, and can be abruptly de-excited from that excited state, is made available for this purpose in the sample being investigated. In STED microscopy, fluorescent dyes are very predominantly used as such substances. In general, the substance is first transferred into the excited state with short-wave light, for example a green laser pulse. The substance is then de-excited in controlled fashion, in a peripheral focus region of the excitation, by means of a long-wave (e.g. red) laser pulse. The de-excitation point function is specifically shaped in order to achieve de-excitation of the substance exclusively in the peripheral focus region. Phase filters are generally used for this purpose; these are located in the beam path of the long-wave laser beam and modify the wavefront of the de-excitation light beam in positionally dependent fashion. What is critical is that the transition from the excited state into the de-excited state, induced by the de-excitation light beam in the peripheral region, take place in saturated fashion, i.e. completely, so that the substance remains in the excited state only in an (in principle, arbitrarily) small central region. The de-excitation light pulse thereby prevents any emission of fluorescent light from the peripheral region of the diffraction-limited excitation spot. The detected fluorescent light thus derives from a narrowly defined sample region whose diameter, because of the saturated de-excitation, can be substantially smaller than allowed by Abbe's law.
Like all other known methods in which an increase in resolution is attained by saturation of a de-excited state, STED microscopy is operated in one-color mode. This means that only one substance, for example only a single fluorescent dye, is made available in the sample. In confocal microscopy, conversely, it is usual to make several different fluorescent dyes available in the sample, and to prepare multi-color images of the sample. It is possible in this fashion to investigate different processes in the sample simultaneously. With multi-color images in the context of conventional microscopy, substantially more information can therefore be obtained (albeit at lower resolution) than is possible with the known spatially high-resolution imaging methods.